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Department of Physiology, University of Kentucky, College of Medicine, Lexington, Kentucky 40536-0098
Address all correspondence and requests for reprints to: Phyllis M. Wise, Ph.D., University of California, Davis, Division of Biological Sciences, 196 Briggs Hall, Davis, California 95616. E-mail: pmwise{at}ucdavis.edu.
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Abstract |
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Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.
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Introduction |
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During the past several years, it has become increasingly clear that neuronal communication depends on neuron-astrocyte and astrocyte-astrocyte interactions. These interactions may play critical roles in the plasticity of GnRH neurons and their ability to communicate cyclically with multiple afferent neurotransmitters. Cyclic morphometric changes in astrocytes during the estrous cycle and following steroid treatment suggest that these cells may play an active role in modulating GnRH neuronal activity. Several studies have shown that glial fibrillary acidic protein (GFAP), an intermediate filament protein and an astrocyte marker, changes over the estrous cycle (3, 4). One of the most intriguing findings is that the surface density of astrocytes in the arcuate nucleus increased on the afternoon of proestrus compared with other times of the cycle (5). The term surface density used by Garcia-Segura et al. (5) is defined as a "quantitative evaluation of the surface density of GFAP-immunoreactive profiles ... in which the ratio of the surface of immunoreactive profiles to the volume of a given structure is calculated by the following formula Sv = 2I/liter." Because changing surface density may allow astrocytes to ensheath neurons to varying extents, astrocytes have been implicated in synaptic plasticity. Indeed, studies have reported alterations in synapses concurrent with alterations in astrocytic apposition and astrocytic volume during the estrous cycle (6, 7). Astrocytes can also affect GnRH secretion via a variety of secreted molecules. Therefore, hypothalamic astrocytes may be necessary instruments in the cyclic release of GnRH.
Age-related changes in the interactions and the diurnal rhythmicity of multiple neurotransmitters and their ability to respond to steroids contribute to the age-related onset of irregular estrous cyclicity (8). In addition, age-related changes in the ability of neurotransmitters to regulate GnRH may contribute to a delay and dampened LH surge during middle age (2). Whether or not alterations in astrocytes contribute to the changing interactions between GnRH and neurotransmitters is unclear. Thus, the purpose of this study was to determine whether 1) the morphometry of astrocytes in the rPOA changes over the estrous cycle; 2) the morphometric changes are unique to astrocytes that are apposed to GnRH neurons; and 3) age influences the plasticity of astrocyte morphometry.
In the present experiment, we assessed the morphometric changes of astrocytes in the rPOA of young and middle-aged regularly cycling females. We reasoned that if cyclic changes occur in astrocyte morphometry and if the changes depend on proximity to GnRH neurons, then astrocytes may play an important role in the cyclic release of GnRH leading to an LH surge.
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Materials and Methods |
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Tissue preparation and dual label immunocytochemistry
Serial 40-µm-thick coronal vibratome sections were collected in six series from the rPOA and stored in cryoprotectant at -20 C until use. Immunocytochemistry was performed on free-floating brain sections from each animal. Sections were washed in Tris-HCl buffer (0.05 M, pH 7.6) to remove the cryoprotectant and incubated in blocking buffer (10% normal horse serum, 0.1% sodium azide, and 0.2% Triton X-100 in Tris-HCl buffer) for 1 h. Sections were then incubated in monoclonal mouse anti-GFAP antibody (Sigma, St. Louis, MO) at a dilution of 1:100,000 in blocking buffer overnight at room temperature. The next day, sections were washed in Tris-HCl, incubated in biotin-conjugated donkey antimouse IgG (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, processed with ABC Elite Standard kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions and stained with nickel-enhanced diaminobenzidine solution [1.5 g nickel ammonium sulfate, 25 mg 3,3'-diaminobenzidine, and 2 µl H2O2/100 ml sodium acetate buffer (0.1 M, pH 6.5)]. Stained sections were washed in Tris-HCl, incubated in blocking buffer for 1 h and then incubated overnight in rabbit anti-GnRH [LR-1, kindly provided by Benoit (Montréal, Canada)] diluted 1:100,000 in blocking buffer overnight at room temperature. The following day, sections were washed in Tris-HCl before exposure to biotin-conjugated donkey antirabbit IgG as described above and stained with diaminobenzidine (50 mg and 5 µl H2O2/100 ml Tris-HCl). Sections were mounted on slides, dried overnight, and coverslipped. GnRH immunoreactivity was identified as a brown precipitate in the cytoplasm, and GFAP immunoreactivity appeared as a black precipitate over the entire astrocyte cell body including the processes.
Microscopy and analyses
Sections containing the rPOA were identified microscopically (x40). GnRH neurons were easily detected and exhibited the classical ovoid morphology. Astrocytes were very abundant and also easy to identify by there amorphous shape with numerous processes extending from the cell body (Fig. 1
). The anatomically matched sections were analyzed using the Bioquant software. Astrocytes were classified as apposed if they appeared to be touching the GnRH neuron or not apposed if they were more than 100 µm from the GnRH neuron (Fig. 2). Using a camera attachment, sections were projected onto a computer monitor and the image was captured. The surface area of individual GFAP immunoreactive cells apposed to and not apposed to GnRH immunoreactive neurons were measured using the drawing tool. Changes in surface area can be attributed to changes in the length and/or number of processes, or changes in the size of the central core of the astrocyte. Briefly, individual astrocytes were carefully traced with a pointer and the computer automatically calculated the total surface area with adjustments made for magnification. Astrocyte number and process number per astrocyte were also measured. Astrocyte number was determined by placing a GnRH neuron in the center of the field of view at x40 magnification, and the number of astrocytes apposed to a GnRH neuron and astrocytes not apposed a GnRH neuron but still within the field of vision was counted using the counting tool. Initially the number of astrocytes needed to provide a representation of rPOA astrocytes was determined. We quantified 10, 20, and 50 astrocytes and found that the variation around the mean was no different for 20 or 50 as it was for 10 (data not shown). Therefore, we quantified 10 astrocytes in the analysis of surface area and process number. Ten astrocytes apposed to a GnRH neuron/section/animal and 10 astrocytes not apposed to a GnRH neuron/section/animal were analyzed for surface area and process number. Averages were taken for each animal. The calculated value for surface area, process number, or cell number at each time point represents the mean for each experimental group.
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0.05 was considered significant for all tests. |
Results |
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with the inset displaying the two cell types of interest, astrocytes and GnRH neurons.
Changes in the surface area of astrocytes in young rats over the estrous cycle
Astrocytes that are directly apposed to GnRH neurons in the rPOA undergo changes in surface area on proestrus in young rats (Figs. 2 and 3). One-way ANOVA revealed a main effect of time of day on the surface area in astrocytes apposed to GnRH neurons [F(3,15) = 10.44; P < 0.0006]. Post hoc analysis showed that astrocyte surface area was highest at 0300 and 0800 h just before the initiation of the GnRHinduced LH surge on proestrus and decreased significantly at 1200 and 1700 h, times when LH concentrations begin to rise and when they peak, respectively (Fig. 3; P < 0.02). The following day, on estrus, surface area returned to levels similar to those seen on proestrous morning and remained at this level on estrus afternoon.
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. One-way ANOVA uncovered a main effect of time of day [F(3,15) = 3.30; P < 0.05]. Over the morning and into the afternoon of proestrus, process number declined. On estrus, the number of processes per astrocyte returned to levels similar to those seen on proestrous morning.
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) [F(3,25) = 1.20; P = 0.34]. The average number of processes per astrocyte also did not change over the time points analyzed (Fig. 5). However, it is important to note that even though the astrocytes not apposed to GnRH neurons did not undergo changes in surface area over the short time intervals assessed on proestrus, two-way ANOVA (time of day x day of the cycle) revealed an effect of the day of the cycle on surface area [F(1,15) = 8.89; P < 0.01]. Post hoc analysis revealed that astrocytes that are not apposed to GnRH neurons display an overall lower surface area on proestrus than on estrus (Fig. 5; P < 0.01).
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. In middle-aged rats, one-way ANOVA revealed that the surface area of astrocytes did not change over the time points analyzed on proestrus (Fig. 6) [F(3,20) = 0.12; P = 0.95].
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; P < 0.007). When comparisons were made between young and middle-age females by two-way ANOVA, there was a main effect of age on astrocyte surface area [F(1,35) = 4.59; P < 0.04]. Post hoc analysis revealed that the surface area of astrocytes on proestrus in middle-aged rats was significantly lower than surface area in young rats (P < 0.04). This low surface area in middle-aged rats was maintained throughout the day and on into estrus such that no cyclic variation in astrocyte surface area over the estrous cycle was detected.
When comparisons were made between young and middle-aged females for the number of processes per astrocyte, two-way ANOVA revealed a main effect of age on process number [F(1,35) = 16.95; P < 0.0002]. Post hoc analysis showed that the number of processes per astrocyte on proestrus for middle-aged rats was lower than that seen for young rats (P < 0.0003).
The number of astrocytes remains constant
Differences across the cycle and with aging were not due to changes in the number of astrocytes in the rPOA. The number of astrocytes apposed to and not apposed to GnRH neurons remained constant throughout the cycle in both young and middle-aged rats. The number of astrocytes in the rPOA was not different in middle-aged rats compared with young rats (Fig. 7).
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Discussion |
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Complex interactions between the brain, anterior pituitary, and gonads regulate estrous cycles and the proper maintenance of these highly orchestrated events is essential in maintaining regular cycles. Any change that alters the synchrony of these events leads to irregularity of cycles. It is important to consider the various interactions among the ensemble of neurotransmitters and neuromodulators secreted by both neurons and astroyctes and the alterations in these normal interactions that may be the underlying cellular mechanism that leads to irregular estrous cycles. The hypothalamus is the main relay station for all the signals that regulate the cyclic secretion of GnRH, which is ultimately the final integrator that communicates all the information to the pituitary gonadotrophs. Therefore, cyclic communication to GnRH neurons must not falter if regular estrous cycles are to be maintained.
We are beginning to appreciate the interactions of neurons and astrocytes in normal signaling events of the central nervous system that are important in the control of puberty, the maintenance of estrous cyclicity, and neuroendocrine aging (10, 11, 12, 13). We chose to focus on astrocytes in the hypothalamus because 1) GnRH neurons are strongly associated with astrocytes (6, 14); 2) during development, astrocytes become sexually differentiated (15); sexual differentiation is associated with changes in axodendritic and axosomatic input that persist in the adult (16, 17); and 3) GFAP mRNA and immunoreactivity show cyclic changes in regularly cycling rodents (3, 4, 5). The present study focused on the rostral preoptic area because this is the area where GnRH neuronal cell bodies reside (18), and prior studies have shown that these neurons are active during the time of the LH surge in both regularly cycling and ovariectomized estrogen-treated rats (1, 19, 20). Our results clearly show that the diurnal pattern of morphometric changes of astrocytes is specific to astrocytes apposed to GnRH neurons and that aging disrupts these morphometric changes.
In young animals, astrocyte surface area and process number decrease before the GnRH-induced LH surge on proestrus before returning to higher levels on estrus. The decrease in astrocyte surface area from 0800–1200 h just before the GnRH surge may decrease astrocytic ensheathment of GnRH perikarya to allow an increase in excitatory inputs. Multiple neurotransmitters and neuropeptides appear to provide stimulatory signals to GnRH neurons on proestrus. Catecholamines, excitatory amino acids, and neurotensin are just a few of the neurosignaling molecules, which have been shown to activate GnRH neurons (21, 22). These stimulatory factors act in concert and/or sequentially to induce a proestrous GnRH-induced LH surge. Therefore, an increase in excitatory inputs on proestrus is necessary to activate GnRH neurons in the rPOA involved in the LH surge.
Prior studies have shown that synaptic input into GnRH neurons changes over the estrous cycle. Specifically in the arcuate nucleus, synaptic input onto GnRH neurons decreased from proestrus to estrus (23). Further studies showed that 
-aminobutyric acid-ergic (GABAergic) input was reduced around the time of the LH surge concurrent with an increase in astrocyte immunoreactive surface density (5, 24). These investigators concluded that the increase in astrocyte surface density would allow astrocytes to ensheath GABAergic neurons and to decrease the number of GABAergic synapses on GnRH neurons. Unlike the changes seen in astrocytes in the arcuate nucleus, astrocytes in the rPOA behave in a different manner. In this study, we demonstrated that astrocytes reduce their surface area around the time of the LH surge possibly to allow for greater synaptic input. In the rPOA, the astrocytes analyzed in this study were surrounding the cell body of the GnRH neuron unlike in the arcuate nucleus where only terminals reside. It is likely that astrocytes in the two areas behave in different manners to regulate the GnRH-induced LH surge. Thus, astrocytic ensheathment of GnRH neurons may modulate synaptic input at critical times during proestrus leading to a regulated GnRH-induced LH surge.
The astrocyte population directly apposed to GnRH neurons appears to be unique. These astrocytes undergo changes in surface area and process number over the estrous cycle, which are not seen in astrocytes that are not apposed to GnRH neurons. The differences seen between these two populations of astrocytes may be interpreted in several ways. The two populations may be derived and differentiate from different functional subpopulations of glia during development. One may migrate to the rPOA, interacting directly with GnRH neurons and the other assuming a more generalized role further away from GnRH neurons. It is interesting to consider such an idea because it is known that GnRH neurons originate in the nasal placode and then migrate to assume their position in the hypothalamus during development and astrocytes provide guidance in the migration of these neurons (25, 26). It remains to be determined whether a certain population of glial cells also migrates with the GnRH neurons destined for the rPOA and then differentiates into mature astrocytes and whether it is these astrocytes that undergo the cyclic morphometric changes during the estrous cycle.
Another possibility is that the local environment influences the astrocytes. In the hypothalamus, multiple neuronal circuits are involved in the regulation of the GnRH surge (21, 22). Different neuronal circuits may influence astrocytes in close apposition to GnRH neurons to cause these astrocytes to express specific receptors or to produce different neuromodulators that are important for astrocyte-GnRH neuron interaction. This possibility seems plausible especially because the controversy surrounding discrete populations of astrocytes remains to be settled (27). It has been argued that astrocytes cannot be divided into distinct and permanent classes based on their expression of specific membrane ion channels or GFAP because these parameters can change when the environment is altered. In fact, in vitro studies have shown that the local environment influences astrocytes (28). Thus, the local environment around GnRH neurons may be different from the environment that is distant from GnRH neurons such that only astrocytes apposed to GnRH neurons undergo morphometric changes on proestrus.
We observed significant attenuation in the plasticity of astrocytes by the time rats are middle age. It is important to point out that we used middle-aged rats that were still cycling regularly but that have been shown to exhibit attenuated and delayed preovulatory LH surges (2). Our results make it clear that declining astrocyte plasticity is an early event in reproductive aging and may drive the alteration in the rhythmicity of neurotransmitters that is required for normal preovulatory and steroid-induced LH surges. We found that middle-aged regularly cycling rats do not show any detectable changes in astrocyte surface area or process number during proestrus. In fact, astrocyte surface area is lower in middle-aged compared with young rats. This would suggest that the suppression of morphometric changes in astrocytes with aging might lead to persistent and acyclic neurotransmitter input into GnRH neurons. We speculate that this unchanging neurotransmitter input may lead to the absence of proper stimulatory tone on proestrous afternoon. Several other age-related changes, such as changes in neurotransmitter turnover rates or receptor density may also contribute to alterations to input to GnRH neurons. Interestingly, we have previously shown that norepinephrine turnover in the medial preoptic nucleus is high on proestrus morning in middle-aged rats (29).
The loss in plasticity may be explained by in vitro studies, which have shown that astrocytes undergo biochemical changes with age (30). The accumulation of oxidative damage may alter normal cellular functions such as the activity of glutamine synthetase, which are important to the astrocytes themselves and to the neurons that rely on astrocytes for neurotransmitter precursors and for stabilization of the extracellular environment. Marchetti et al. (13) have shown that coculturing young glia with GnRH-like GT1-1 cells induces the release of GnRH, whereas coculturing GT1-1 cells with old glia does not induce release. Therefore, aging could lead to a loss of cellular maintenance and/or the expression of certain proteins that are needed for astrocytes to interact with GnRH neurons.
Multiple studies have shown age-related changes in the interactions and the diurnal rhythmicity of numerous neurotransmitters and their receptors involved in the regulation of regular estrous cycles. The question how these complex interactions occur and how different cell types, neurons and/or astrocytes interact remains open. Neurotransmitters may be acting on astrocytes apposed to GnRH neurons or on GnRH neurons directly. Astrocytes express many receptors for neurotransmitters such as metabotrophic glutamate receptors and GABA receptors (31, 32). An active partnership between astrocytes and GnRH neurons in the hypothalamus to integrate all the various neural inputs must be investigated. The role of astrocytes in the hypothalamic events leading to the GnRH-induced LH surge, therefore, requires further examination.
In summary, findings from this study and future studies will provide new insights into the regulation of GnRH neurons, which may help in better understanding the physiological and cellular mechanisms that lead to reproductive senescence.
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Footnotes |
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Abbreviations: GABAergic, -Aminobutyric acid-ergic; GFAP, glial fibrillary acidic protein; ir, immunoreactive; rPOA, rostral preoptic area.
Received July 12, 2002.
Accepted for publication September 6, 2002.
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